Project Summary/Abstract The World Health Organization reports diarrhea kills around 760,000 children under five every year. Cryptosporidium is a leading cause of diarrhea morbidity and mortality. Therefore novel treatments against this pathogen are urgently needed. The limitations of tools used to genetically manipulate gene expression in this parasite have been identified as a major hurdle for drug and vaccine development. To circumvent this gap, we developed a rapid method to silence genes in this parasite using pre-assembled complexes (minimal RISC complexes, ready to silence) of Cryptosporidium single-stranded RNA and the human enzyme Argonaute 2 (ssRNA/Ago). We used this method to determine the role of selected genes during Cryptosporidium infection. We identified four genes (Actin, Nucleoside diphosphate kinase, Rhomboid protein 1 and transcription factor Ap2) that block proliferation and egress of Cryptosporidium parasites. Therefore, we hypothesized that ssRNA/Ago based therapy may be optimized in order to treat Cryptosporidium infection. In this project our goal is to develop a method to block parasite proliferation on infected intestinal cells. Our specific aims are: 1) Developing a novel method to reduce Cryptosporidium infection by silencing essential genes and to test the silencing with ssRNA/Ago complexes under physiological conditions and 2) Demonstrate the feasibility to deliver ssRNA-Ago complexes in the intestine. The characterization of these mechanisms will be essential to explore the feasibility to use RNA interference technology as novel alternative for the treatment of Crypotosporidiosis